I.P. Perpétuo

Cytokine pattern in very early rheumatoid arthritis favours B-cell activation and survival

OBJECTIVES B cells play an important role in the perpetuation of RA, particularly as autoantibody-producing cells. The ICs that further develop deposit in the joints and aggravate the inflammatory process. However, B-cell contribution in the very early stage of the disease remains unknown. The main goal of this work was to determine the concentration of cytokines potentially relevant for B-cell activation in serum from very early polyarthritis patients, with <6 weeks of disease duration, who latter on evolved into very early RA (VERA). METHODS A proliferation-inducing ligand (APRIL), B-cell activating factor (BAFF) and IL-21 levels were measured by ELISA in the serum of VERA, other very early arthritis (VEA), established RA patients and controls. SF samples of established RA were also analysed. RESULTS VERA patients have higher levels of APRIL and BAFF as compared with VEA, established RA and controls. Furthermore, APRIL and BAFF levels are also significantly elevated in RA-SF when compared with serum. CONCLUSIONS The increased levels of APRIL and BAFF in VERA patients suggests that B-cell activation and the development of autoreactive B-cell responses might be crucial in early phases of RA. Therefore, APRIL and BAFF could be promising targets for therapy in the early phase of RA.

Upregulation of Inflammatory Genes and Downregulation of Sclerostin Gene Expression Are Key Elements in the Early Phase of Fragility Fracture Healing

Background Fracture healing is orchestrated by a specific set of events that culminates in the repair of bone and reachievement of its biomechanical properties. The aim of our work was to study the sequence of gene expression events involved in inflammation and bone remodeling occurring in the early phases of callus formation in osteoporotic patients. Methodology/Principal Findings Fifty-six patients submitted to hip replacement surgery after a low-energy hip fracture were enrolled in this study. The patients were grouped according to the time interval between fracture and surgery: bone collected within 3 days after fracture (n = 13); between the 4th and 7th day (n = 33); and after one week from the fracture (n = 10). Inflammation- and bone metabolism-related genes were assessed at the fracture site. The expression of pro-inflammatory cytokines was increased in the first days after fracture. The genes responsible for bone formation and resorption were upregulated one week after fracture. The increase in RANKL expression occurred just before that, between the 4th–7th days after fracture. Sclerostin expression diminished during the first days after fracture. Conclusions The expression of inflammation-related genes, especially IL-6, is highest at the very first days after fracture but from day 4 onwards there is a shift towards bone remodeling genes, suggesting that the inflammatory phase triggers bone healing. We propose that an initial inflammatory stimulus and a decrease in sclerostin-related effects are the key components in fracture healing. In osteoporotic patients, cellular machinery seems to adequately react to the inflammatory stimulus, therefore local promotion of these events might constitute a promising medical intervention to accelerate fracture healing.

Chronic arthritis leads to disturbances in the bone collagen network

IntroductionIn this study we used a mice model of chronic arthritis to evaluate if bone fragility induced by chronic inflammation is associated with an imbalance in bone turnover and also a disorganization of the bone type I collagen network.MethodsSerum, vertebrae and femur bones were collected from eight-month-old polyarthritis SKG mice and controls. Strength of the femoral bones was evaluated using three-point bending tests and density was assessed with a pycnometer. Bone turnover markers carboxy-terminal collagen cross-linking telopeptides (CTX-I) and amino-terminal propeptide of type I procollagen (PINP) were measured in serum. The organization and density of bone collagen were analyzed in vertebrae using second-harmonic generation (SHG) imaging with a two-photon microscope and trabecular bone microstructure was assessed by scanning electron microscope (SEM).ResultsFemoral bones of SKG mice revealed increased fragility expressed by deterioration of mechanical properties, namely altered stiffness (P = 0.007) and reduced strength (P = 0.006), when compared to controls. Accordingly, inter-trabecular distance and trabecular thickness as observed by SEM were reduced in SKG mice. PINP was significantly higher in arthritic mice (9.18 ± 3.21 ng/ml) when compared to controls (1.71 ± 0.53 ng/ml, P < 0.001). Bone resorption marker CTX-I was 9.67 ± 3.18 ng/ml in arthritic SKG mice compared to 6.23 ± 4.11 ng/ml in controls (P = 0.176). The forward-to-backward signal ratio measured by SHG was higher in SKG animals, reflecting disorganized matrix and loose collagen structure, compared to controls.ConclusionsWe have shown for the first time that chronic arthritis by itself impairs bone matrix architecture, probably due to disturbed bone remodeling and increased collagen turnover. This effect might predispose patients to bone fragility fractures.

Rheumatoid Arthritis Bone Fragility Is Associated With Upregulation of IL17 and DKK1 Gene Expression

Our aim was to compare bone gene expression in rheumatoid arthritis (RA) and primary osteoporosis (OP) patients. Secondary aims were to determine the association of gene expression of the Wnt/β-catenin signaling pathway with inflammatory cytokines in the bone microenvironment and to assess the serum levels of Wnt/β-catenin proteins in both groups. RA patients referred for hip replacement surgery were recruited. Primary OP patients were used as controls. Gene expression of Wnt pathway mediators, matrix proteins, and pro-inflammatory cytokines were analyzed in bone samples. Bone turnover markers, inflammatory cytokines, and Wnt mediators were measured in serum. Twenty-two patients were included: 10 with RA and 12 with primary OP. The expressions of Wnt10b (p = 0.034), its co-receptor LRP6 (p = 0.041), and its negative regulator DKK1 (p = 0.008) were upregulated in RA bone. IL17 gene expression in bone was upregulated in RA patients (p = 0.031) and correlated positively with Wnt10b (r = 0.810, p = 0.015), DKK2 (r = 0.800, p = 0.010), and RANKL/OPG ratio (r = 0.762, p = 0.028). DKK2 (p = 0.04) was significantly decreased in RA serum compared with primary OP. In conclusion, bone fragility in RA patients is induced by an unbalanced bone microenvironment and is associated with a specific gene expression pattern, namely, the upregulation of IL17 and DKK1, suggesting that the modulation of these two pathways might prevent RA systemic bone loss.

Low osteocalcin/collagen type I bone gene expression ratio is associated with hip fragility fractures

INTRODUCTION Osteocalcin (OC) is the most abundant non-collagenous bone protein and is determinant for bone mineralization. We aimed to compare OC bone expression and serum factors related to its carboxylation in hip fragility fracture and osteoarthritis patients. We also aimed to identify which of these factors were associated with worse mechanical behavior and with the hip fracture event. METHODS In this case-control study, fragility fracture patients submitted to hip replacement surgery were evaluated and compared to a group of osteoarthritis patients submitted to the same procedure. Fasting blood samples were collected to assess apolipoproteinE (apoE) levels, total OC and undercarboxylated osteocalcin (ucOC), vitamin K, LDL cholesterol, triglycerides and bone turnover markers. The frequency of the apoε4 isoform was determined. Femoral epiphyses were collected and trabecular bone cylinders drilled in order to perform compression mechanical tests. Gene expression of bone matrix components was assessed by quantitative RT-PCR analysis. RESULTS 64 patients, 25 submitted to hip replacement surgery due to fragility fracture and 39 due to osteoarthritis, were evaluated. Bone OC/collagen expression (OC/COL1A1) ratio was significantly lower in hip fracture compared to osteoarthritis patients (p<0.017) adjusted for age, gender and body mass index. Moreover, OC/COL1A1 expression ratio was associated with the hip fracture event (OR ~0; p=0.003) independently of the group assigned, or the clinical characteristics. Apoε4 isoform was more frequent in the hip fracture group (p=0.029). ucOC levels were higher in the fracture group although not significantly (p=0.058). No differences were found regarding total OC (p=0.602), apoE (p=0.467) and Vitamin K (p=0.371). In hip fracture patients, multivariate analysis, adjusted for clinical characteristics, serum factors related to OC metabolism and gene expression of bone matrix proteins showed that low OC/COL1A1 expression ratio was significantly associated with worse trabecular strength (β=0.607; p=0.013) and stiffness (β=0.693; p=0.003). No association was found between ucOC and bone mechanics. Moreover, in osteoarthritis patients, the multivariate analysis revealed that serum total OC was negatively associated with strength (β=-0.411; p=0.030) and stiffness (β=-0.487; p=0.009). CONCLUSION We demonstrated that low bone OC/COL1A1 expression ratio was an independent predictor of worse trabecular mechanical behavior and of the hip fracture event. These findings suggest that in hip fracture patients the imbalance of bone OC/COL1A1 expression ratio reflects disturbances in osteoblast activity leading to bone fragility.

Cytokine pattern in very early rheumatoid arthritis favours B cell activation and survival

B cells play an important role in the perpetuation of rheumatoid arthritis (RA), particularly as autoantibody producing cells. The immune complexes that further develop deposit in the joints and aggravate the inflammatory process. However, B cells contribution in the very early stage of the disease remains unknown.

Lymphotoxin-α 252 A>G Polymorphism: A Link Between Disease Susceptibility and Dyslipidemia in Rheumatoid Arthritis?

Objective. Rheumatoid arthritis (RA) is associated with higher levels of inflammatory mediators and with a more atherogenic lipid profile. Dyslipidemia can be present years before arthritis develops. Lymphotoxin-α (LTA) is a cytokine that mediates proinflammatory responses while also participating in lipid homeostasis, and its transcriptional activity is in part genetically determined. We examined the role of the single-nucleotide polymorphism at position 252 of the LTA gene in the genetic background of RA and dyslipidemia. Methods. The association between the LTA 252 A>G polymorphism and disease status was examined in a nested case-control study of 388 patients with RA and 269 unrelated healthy controls, all white. Demographics and disease features were assessed, fasting lipids measured, and the use of lipid-lowering agents evaluated. Results. The LTA 252 A allele was more frequent in cases compared to controls (70.5% and 64.3%, respectively; p = 0.018, OR 1.325, 95% CI 1.049–1.675), as well as the A/A genotype (50.8% vs 43.5%; p = 0.025). The A/A genotype was independently associated with dyslipidemia in patients, but not in controls. Patients with RA who had the LTA 252 G/G genotype were younger at disease onset and had higher C-reactive protein (CRP) levels. Conclusion. We found the LTA 252 A allele to be associated with an increased risk for developing RA in whites. The LTA 252 A/A genotype translates to a phenotype more prone to dyslipidemia, and the G/G genotype to a phenotype with earlier onset of disease and higher levels of CRP, when RA does occur. These observations highlight a possible common genetic predisposition to RA and dyslipidemia.

Pachydermoperiostosis in an African patient caused by a Chinese/Japanese SLCO2A1 mutation—Case report and review of literature

OBJECTIVES Pachydermoperiostosis is a rare clinical entity characterized by skin thickening of the forehead, eyelids, and hands, digital clubbing, and periostosis. Two genes have been associated, HPGD and recently SLCO2A1. We present a detailed clinical and genetic description of an African pachydermoperiostosis patient with a SLCO2A1 mutation. METHODS Standard clinical and laboratory evaluation was carried out. Genetic screening was done with PCR followed by direct sequencing. We discuss the clinical features and known mutations of previously reported cases identified through a PubMed literature review. RESULTS The clinical findings showed special features, including exuberant knee effusions and an extraordinary good response on surgery of the blepharoptosis. We found a splice site mutation in the SLCO2A1 gene in homozygous form: c.940+1G>A. This mutation was previously reported only in 1 Chinese and 3 Japanese cases and was considered as a founder mutation in Japan. Beside our case, only one other patient in the literature carried this mutation in homozygous condition, but with different main clinical symptoms. CONCLUSIONS Our case demonstrates phenotypic heterogeneity of PDP even between homozygous carriers of the same mutation, suggesting further modifiers. Besides, it shows that this rare SLCO2A1 mutation is not exclusively present in East-Asia, but can occur in various ethnicities, with different origin, thus the incidence is probably underestimated.

Effect of Tumor Necrosis Factor Inhibitor Therapy on Osteoclasts Precursors in Ankylosing Spondylitis.

Introduction Ankylosing Spondylitis (AS) is characterized by excessive local bone formation and concomitant systemic bone loss. Tumor necrosis factor (TNF) plays a central role in the inflammation of axial skeleton and enthesis of AS patients. Despite reduction of inflammation and systemic bone loss, AS patients treated with TNF inhibitors (TNFi) have ongoing local bone formation. The aim of this study was to assess the effect of TNFi in the differentiation and activity of osteoclasts (OC) in AS patients. Methods 13 AS patients treated with TNFi were analyzed at baseline and after a minimum follow-up period of 6 months. 25 healthy donors were recruited as controls. Blood samples were collected to assess receptor activator of nuclear factor kappa-B ligand (RANKL) surface expression on circulating leukocytes and frequency and phenotype of monocyte subpopulations. Quantification of serum levels of bone turnover markers and cytokines, in vitro OC differentiation assay and qRT-PCR for OC specific genes were performed. Results RANKL+ circulating lymphocytes (B and T cells) and IL-17A, IL-23 and TGF-β levels were decreased after TNFi treatment. We found no differences in the frequency of the different monocyte subpopulations, however, we found decreased expression of CCR2 and increased expression of CD62L after TNFi treatment. OC number was reduced in patients at baseline when compared to controls. OC specific gene expression was reduced in circulating OC precursors after TNFi treatment. However, when cultured in OC differentiating conditions, OC precursors from AS TNFi-treated patients showed increased activity as compared to baseline. Conclusion In AS patients, TNFi treatment reduces systemic pro osteoclastogenic stimuli. However, OC precursors from AS patients exposed to TNFi therapy have increased in vitro activity in response to osteoclastogenic stimuli.

Ankylosing Spondylitis Patients Have Impaired Osteoclast Gene Expression in Circulating Osteoclast Precursors.

[This corrects the article on p. 5 in vol. 4, PMID: 28191455.].